Method of Preparing Enriched Antibodies for Detecting Mycobacterial Infection

ABSTRACT

The disclosed technology provides an enriched antibody population, highly specific for an antigen of a surface polysaccharide, from a mycobacterium. In a related embodiment, the antibody is enriched by having been raised in an environment that maintains antigenically active antigen. These antibodies may be used in an immunoreactive environment for detecting the presence of a mycobacterial infection in a sample from a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/932,722, filed Oct. 31, 2007 (hereby incorporated herein byreference), which is a divisional application of U.S. application Ser.No. 11/186,933, filed Jul. 20, 2005 (hereby incorporated herein byreference), now U.S. Pat. No. 7,335,480, which claims the benefit ofU.S. Provisional Application No. 60/589,419, filed Jul. 20, 2004 (herebyincorporated herein by reference).

TECHNICAL FIELD

The present invention relates to diagnostic tests for detectingmicrobial-based diseases and conditions, and more particularly fordiagnostic tests and methods for detecting tuberculosis.

BACKGROUND

During the last decades TB has evolved from a predominantly pulmonaryinfection into a multifaceted pathology with a growing rate ofextrapulmonary cases. Until to date effective TB prevention programs arehampered by the absence of a rapid and field adapted screening assay. Inhigh-income countries mycobacterial culture remains the diagnosticstandard, but it is time-consuming and relatively expensive. Ideally,sputum microscopy based on three sputum smears can identify up to 67% ofculture positive cases. HIV co-infection has been reported to impair thedemonstration of Mycobacterium tuberculosis in sputa, although someinvestigators do not report any influence of the HIV serostatus on theAFB diagnosis. The higher percentage of extrapulmonary TB in HIVpositive TB patients additionally increases the rate of AFB-negative TBcases. This renders tuberculosis an increasing diagnostic challenge andunderlines an urgent need for improved laboratory tools for itsdiagnosis.

Current approaches for diagnosing TB are not satisfactory. The sputumtest for pulmonary TB is not always effective, particularly if there areno detectable bacteria in the sputum, or no sputum sample can beobtained. In addition, this diagnostic test requires microscopy and/orculture of the bacteria to confirm the diagnosis, neither of which isespecially suitable to diagnosis in the field. cerebrospinal fluid fordiagnosis of TB-meningitis is also problematic, particularly in thefield since, once again, microscopy and/or culture of the bacteriaand/or an ELISA test is usually required to confirm the diagnosis.

Blood tests for TB are also known, but have a poor track record, beingcomplex and unreliable. Urine tests are simpler and more reliable, butcurrent methods require processing of the urine before performing thediagnostic test—such processing usually involving concentration of theurine.

Among the newly developed methods antibody tests against a number ofmycobacterial antigens have been developed, but none of these tests hasso far reached the needed specificity for routine diagnostic purpose.The drop of sensitivity in HIV positive cases is also a majorconstraint. A different approach is to measure immune responses toMycobacterium tuberculosis specific antigens like ESAT-6, but so far thedifferentiation between latent TB infection and TB disease is notpossible.

Tuberculosis is an extremely complex pathology existing in multipleforms, but always starting as an airborne infection. Pulmonarytuberculosis occurs immediately at the entry point of the microorganismand extrapulmonary tuberculosis is the result of further penetrationinto the body of the patient with the most widespread examples oftuberculous meningitis and bone tuberculosis. Complexity of thepathology determines multitude of various approaches tried during thiscentury of modern medicine. Furthermore clinical and radiographicmanifestations of HIV-related pulmonary tuberculosis are dramaticallyaltered by immunodeficiency. These factors severely limit our capabilityof early symptomatic recognition of tuberculosis in HIV/TB patients andalso increase the danger of TB transmission to relatives and caregiversof such patients.

Mycobacteria can potentially be recovered from a variety of clinicalspecimens, including upper respiratory collections (sputum, bronchialwashes, bronchoalveolar lavage, bronchial biopsies and such); urine,feces, blood, cerebrospinal fluid (CSF), tissue biopsies, and deepneedle aspirations of virtually any tissue or organ. Bacterial cultureremains the gold standard in the diagnosis of tuberculosis, but it cantake up to 6-8 weeks to make a conclusive diagnosis. There are threemajor technologies used for rapid (faster than bacterial culture)diagnosis of the mycobacterial infections:

Direct microscopy of sputum smears;

PCR-based assays;

Immunodiagnostic methods.

Direct microscopy of sputum smears. More than a century ago, Robert Kochidentified the etiologic agent of tuberculosis by staining it andculturing it from clinical specimens. Today, the diagnosis oftuberculosis is usually established using staining and culturingtechniques that do not differ substantially from those that Koch used.Direct microscopy of sputum is the norm for the diagnosis oftuberculosis in developing countries today and it is the benchmarkagainst which the efficiency of any new test must be assessed. It isapplied to pulmonary tuberculosis, but is not very useful for childrenor for patients with initial stages of pulmonary tuberculosis.

PCR-based assays. A comparative study of the performance of PCR tests inseven laboratories has shown high levels of false-positive PCR-results,ranging from 3% to 20% (with an extreme of 77% in one laboratory). Thisrelatively poor performance resulted from lack of monitoring of eachstep of the procedure and underscores the necessity for careful qualitycontrol during all stages of the assay.

Immunodiagnostic Methods.

The Tuberculosis Skin Test. This is the probably oldest immunologicaltest for tuberculosis. A small amount of substance called PPD Tuberculinis placed just under the top layer of the skin on the forearm with asmall needle. The test is read 48 to 72 hours after it has been given.Generally, a swelling of 10 mm. or more is considered positive. Manydeveloping countries use BCG vaccination to protect against TB. AfterBCG vaccination, the PPD skin test usually becomes positive. Results ofthe skin test vary dependent on the quality of the PPD antigen,reactivity of the immune system and probably even race of theindividual. This test also does not provide an unequivocal indicationabout the stage and location of the infection.

Serological tests for M. tuberculosis. This approach, based on thedetection of antibody immune response to mycobacterial antigens is oneof the most widely used in research and clinical environments. Allserological tests have approximately the same sensitivity andspecificity if they use purified antigens. The sensitivity of the besttests is in a range of 80% for smear-positive cases and 60-70% for smearnegative cases. The reported specificity is generally high and is in arange of 95-100%.

Currently existing technologies are limited in their performance inseveral ways.

Most widely accepted rapid microscopic test requires several hours tocomplete, skillful technician and clinical laboratory environment. Testinterpretation is far too difficult compared to current standards ofrapid POC (point of care) testing in the infectious diseases area. Realcost of one analysis per one patient runs in the range of $100-150 for aUS hospital. Clinical specificity of the test is very good, but anyimprovements in sensitivity will be more than welcome.

Skin test has sufficient sensitivity, but takes a long time and does notprovide information about stage of pathological process and does notsufficiently differentiate infected and vaccinated individuals.

Serological tests usually do not have sufficient sensitivity. Testresults vary with variations in the individual immune response to TBantigens. These tests practically do not work in HIV patients infectedby M. tuberculosis. This factor severely limits their applicability inAfrica and many Asian countries. In the US this group of patientsconstitutes the majority of TB infected patients as well.

PCR tests are widely used in developed countries, but are complex,expensive and are not sensitive enough to justify their use as ascreening test in developing countries.

A preferred method for rapid diagnosis of infectious diseases is basedon the detection of a bacterial antigen in the patient sample, thatprovides unequivocal proof of active infectious process caused byspecific pathogen. The concept of using a direct antigen test fordetection of mycobacterial infections was described in severalpublications.

For example, the development of one of the first direct antigen assayfor M. tuberculosis was reported in 1982—a radioimmunoassay for thedetection M. tuberculosis antigens in sputum of patients with activepulmonary tuberculosis, using a rabbit antibody specific to the wholecells of M. Bovis (BCG vaccine). Autoclaved and sonicated sputum wasused as a sample. The assay detected antigen in 38 of 39 sputum samplesfrom patients with active tuberculosis pulmonary tuberculosis.

Later studies reported the development of the ELISA system for thedetection of mycobacterial antigens in the cerebrospinal fluid ofpatients with tuberculous meningitis, also using antibodies specific tothe whole cells of M. bovis. Both systems showed surprisingly highspecificity. Despite the fact that LAM was the major antigen responsiblefor the detection, it was reported that M. kansasii showed 5%cross-reactivity, and M. intracellulare, M. avium, M. fortitum, and M.vaccae cross-reacted only at 2%. Others reported detection, by ELISA, ofmycobacterial antigen in the CSF of nine of 12 patients with tuberculousmeningitis, corresponding to the sensitivity of 81.25%. Specificity ofthe test was equal to 95%.

Practically all previous attempts to develop a test for diagnosis oftuberculosis have focused on the detection of the pulmonary form of thedisease. Extrapulmonary forms, which are notoriously difficult todiagnose, attracted relatively little attention due to low prevalencerate compared to pulmonary forms. Until the 1950s and 1960s,extrapulmonary TB cases comprised only around 10% of all tuberculosiscases. The onset of the HIV/AIDS pandemic has changed the situationcompletely. These two diseases eventually merged into a new complexpublic health problem. Now fully 60% of untreated HIV patients developactive TB during their lifetime and up to 70% of TB patients are HIVinfected in sub-Saharan Africa and Asia. Superimposition of HIV and TBchanged not only the epidemiology of tuberculosis, but also the courseof the disease itself. During the last decades TB has evolved frompredominantly a pulmonary infection into a multifaceted pathology withan ever growing prevalence of extrapulmonary forms. It is estimated thatextrapulmonary TB cases currently comprise up to 30% of all cases oftuberculosis; this number might even be an underestimation due to thelack of tools for rapid screening and diagnosis of extrapulmonary formsof tuberculosis. Moreover, even pulmonary tuberculosis in HIV patientsfrequently exhibits atypical symptoms. For example, such patientstypically do not produce sputum. These factors severely limit ourcapability of early symptomatic recognition of tuberculosis in HIV/TBpatients and also increase the danger of TB transmission to relativesand caregivers of such patients. An easy to use screening test, capableof detecting a broad spectrum of pathologies due to M. tuberculosisinfection, is urgently needed, including a test for extrapulmonary formsof TB. Such a need has long been discussed with no progress towardsrealising goal. Today the need has became a public health careemergency.

In other cases of pulmonary bacterial infections, the current screeningprocess of choice is based on the detection of polysaccharide antigenssecreted in the patient's urine. Bacterial polysaccharides are composedof monosaccharides uncommon to humans and therefore resistant tocleavage by human enzymes. This enables their secretion in urine inimmunochemically intact forms suitable for detection by apolysaccharide-specific immunoassay. Extremely low concentrations ofbacterial polysaccharides secreted in urine require very highsensitivity of the immunoassay in order to use it as a screeningprocedure.

Collaborating research groups from Sweden and Norway attempteddevelopment of a LAM-specific ELISA system detecting LAM antigen inpatient urine. The system used antigen capture for detectingtuberculosis from urine based on lipoarabinomannan, a polysaccharidepresent on the surface of Mycobacterium tuberculosis, the organismresponsible for causing tuberculosis in humans, as disclosed in PCTapplication no. WO97/34149 to Svenson, hereby incorporated by referenceherein. The disclosed diagnostic procedure detected the presence of LAMin patient urine in 81.3% of AFB-positive patients and 57.4% ofAFB-negative patients and demonstrated utility of the detection ofmycobacterial LAM antigen for diagnosis of mycobacterial infections. Atthe same time the system failed to demonstrate utility of the disclosedprocess for screening purposes. Despite use of the affinity purifiedrabbit polyclonal antibody specific to LAM antigen, the procedure lackedsufficient sensitivity to be used on non-processed un-concentrated urinesamples. The diagnostic procedure required approximately 24-48 hrs ofsophisticated manipulations in a biochemical lab focused onconcentrating patient urine and preparing it for analysis by ELISA test.Overall, the sensitivity of the Svenson assay is not sufficient forpractical use of the disclosed method. The complexity and length of theimmunoassay also prevents its practical use as a screening test fordetection of mycobacterial infections because it proved too cumbersomefor use in a clinical setting, where speed, ease of use, and highsensitivity are all critically important for diagnostic tests used todetect disease conditions.

SUMMARY OF THE INVENTION

In a first embodiment of the invention there is provided anantigenically active isoform of lipoarabinomannan from mycobacteriumtuberculosis, prepared by oxidation of LAM using mild oxidation methodssuch as treatment with low concentrations of NaIO₄. In otherembodiments, the antigenically active isoform of LAM, generated by mildoxidation methods, is used to prepare highly specific, highly pureantibodies to inactivated mycobacterium, more particularly to surfacepolysaccharides such as LAM, for use in the detection of polysaccharides(e.g. LAM) in urine, sputum, blood, tissue or other samples frompatients of interest. Other embodiments use the highly specific, highlypure antibody raised to the antigenically active form of LAM to diagnosetuberculosis in patients of interest.

In another particular embodiment, there is provided an enriched antibodypopulation highly specific for an antigen of a surface polysaccharidefrom a mycobacterium. In this embodiment, the enriched antibodypopulation may be enriched by having been raised in an environment thatmaintains antigenically active antigen. Alternatively or in addition,the antibody is enriched by exclusion of antibodies that recognizerelatively inactive antigen. In some embodiments, the mycobacterium maybe Mycobacterium tuberculosis. Similarly, the surface polysaccharide maybe lipoarabinomannan (LAM).

In another embodiment, there is provided a process for producing anenriched antibody highly specific to an antigen of a mycobacterium. Inthis embodiment, the process comprises raising and isolating antibody toantigen from mycobacteria; and separating from the isolated antibodiesthat population of antibodies which is specific to relatively inactiveantigen to produce isolated enriched antibody.

In another embodiment, there is provided a process for producing anenriched antibody highly specific to an antigen of a mycobacterium. Inthis embodiment, the process comprises isolating antigen frommycobacteria under conditions maintaining antigenic activity; andraising antibodies to the isolated antigen while maintaining itsantigenic activity.

In still another embodiment, there is provided a process for producingan enriched antibody highly specific to an antigen of a mycobacterium.In this embodiment, the process comprises applying sera from a mammalinoculated with mycobacteria to a first affinity matrix prepared withisolated antigen from mycobacterium such that antibody specific to theisolated antigen is retained by the first affinity matrix; isolatingantibody specific to the isolated antigen from the first affinitymatrix; applying the isolated antibody to a second affinity matrixprepared with modified antigen from mycobacterium such that antibodyspecific to the modified antigen is retained by the second affinitymatrix, wherein the modified antigen has been treated with an agent todeactivate it relative to the isolated antigen; isolating enrichedantibody specific to the isolated antigen by collecting effluent fromthe second affinity matrix, so that the enriched antibody is more highlyspecific and displays higher sensitivity to mycobacterium antigen thannon-enriched antibody.

In further related embodiments, the mycobacterium may be Mycobacteriumtuberculosis. and the surface polysaccharide may be lipoarabinomannan(LAM).

In other embodiments, the agent for modifying the antigen frommycobacterium is sodium periodate. In other related embodiments, thesurface polysaccharide may be isolated from Freund's adjuvant.

Still another embodiment provides a method for detecting a mycobacterialinfection in a sample from a subject. In this embodiment, the methodcomprises providing an immunoreactive environment, such environmentdeveloped from the enriched antibody as described above; and reactingthe sample in the immunoreactive environment so as to detect themycobacterial infection.

Optionally the mycobacterial infection may be M. tuberculosis or Johne'sdisease. Similarly, the surface polysaccharide may be lipoarabinomannan(LAM). In further related embodiments, the immunoreactive environmentcomprises an ELISA, and may be implemented as a strip test. In relatedembodiments, the mycobacterial infection may be a pulmonaryMycobacterium tuberculosis infection or an extra-pulmonary Mycobacteriumtuberculosis infection, and the sample may be any of sputum, blood,urine, tissue or other suitable sample. In a related particularembodiment, the sample may be non-processed unconcentrated urine.

Other embodiments provide a kit for detecting a mycobacterial infectionin a sample, the kit comprising an assay providing an immunoreactiveenvironment wherein the environment comprises an enriched antibody asdescribed above. In related embodiments, the immunoreactive environmentcomprises an ELISA, and may be implemented as a strip test. In furtherrelated embodiments, the mycobacterial infection may be Mycobacteriumtuberculosis and the surface polysaccharide may be lipoarabinomannan(LAM).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features of the invention will be more readily understoodby reference to the following detailed description, taken with referenceto the accompanying drawings, in which:

FIGS. 1A and 1B show a structural model of mycobacterial ManLAM, PILAM,and AraLam. Structural model of mycobacterial ManLAM, PILAM, and AraLAM.MTP corresponds to 5-methylthiopentose described up to date (*) in M.tuburculosis strains H37Rx, H37Ra, CSU20 and MT K3. 5′ corresponds tosuccinyl residues located on arabinan domain of ManLAM of M. bovis BCG.One to four succinyl groups, depending on the M. bovis BCG strain, wereshown to esterify the 3,5-α-Araf units at position 0-2. MPT,mannosyl-phosphotidyl-myo-inositol; Manp, mannopyranose; Araf,arabinofuranose; Ins, myo-inositol; R_(a), fatty acyl groups. ManLAMcontain approximately 60 Araf and 40 Manp units. Manp units aredistributed among the mannose caps and the mannan core.

FIG. 2 shows a comparison of serological activity for LAM experiments.

FIG. 3 shows the efficiency of LAM-specific Ab preparations in captureELISA.

FIG. 4A Sensitivity of the LAM ELISA for different concentrations of LAMin urine. Solid circles represent ELISA results using LAM from M.tuberculosis, and open circles represent the control ELISA results. Thecut off was the Optical Density of the Negative Control+0.1, resultingin a minimal detection limit of 0.25 ng/ml.

FIG. 4B Binding of LAM-specific antibodies in the ELISA tonon-mycobacterial antigens was excluded for the following bacterialspecies:

Klebsiella pneumoniae, Streptococcus agalactiae, Streptococcuspneumoniae 14/12F, Pseudomonas aeruginosa, Staphylococcus aureus25923/43300, Proteus vulgaris, E. coli 8739, Neisseria meningitidisA/B/13102, Haemophilus influenzae A/B/D. Open triangles represent ELISAresults for binding of LAM-specific antibodies to antigens of M.tuberculosis, whereas the remaining symbols (e.g. solid triangles, openand solid diamonds, open and solid circles.) represent ELISA resultsusing the same LAM-specific antibodies with other bacterial speciesdescribed above.

FIG. 4C Sensitivity of the LAM ELISA for various mycobacterial strains.LAM of M. bovis and M. tuberculosis are detected most sensitively.

FIG. 5 Correlation between the microscopic mycobacterial density of AFBpositive patients and their antigen concentration measured by the LAMELISA in unprocessed urine. AFB+ (light microscopy 1000× magnification:4-90 acid fast bacilli/100 fields) 28 cases. AFB++ (1-9/field) 23 cases.AFB+++ (˜10/field) 20 cases. Box plot showing 10^(th), 25^(th), 50^(th),75^(th), 90^(th) percentile and the mean antigen concentration.

FIG. 6 shows a schematic of an antigen purification process inaccordance with particular embodiments of the claimed invention.

FIG. 7 shows a schematic for preparing affinity columns in accordancewith particular embodiments of the claimed invention.

FIG. 8 shows a schematic of an antibody purification process inaccordance with particular embodiments of the present invention.

FIG. 9 shows a schematic of a conjugate preparation, in accordance withparticular embodiments of the present invention.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS Definitions

The following terms shall have the meanings indicated unless the contextotherwise requires:

“Immunoreactive environment” as used herein means, an environmentsupportive of immunoassays, immunoreactions, immunochemistry, and anyprocess, assay, methodology or system which involves, relates to orrelies on an immunological reaction to achieve a desired result.Examples of immunoreactive environments are those detailed in U.S. Pat.No. 5,073,484 to Swanson et al.; and U.S. Pat. Nos. 5,654,162 and6,020,147 to Guire et al, incorporated by reference herein, disclosingmethod and apparatus for quantitatively determining an analyte in aliquid, wherein particular embodiments employ immunochemical reactionsin which the analyte and the reactant represent different parts of aspecific ligand (antigen)-antibody (anti-ligand) binding pair. Thesepatents relate to technology that has been implemented as what we callin this description and the following claims as a “strip test.”

“Freund's adjuvant” is from Sigma, USA.

We have developed a high-sensitivity method for detecting the presenceof mycobacterium antigens, particularly M. tuberculosis antigens, suchas the surface polysaccharides lipoarabinomannan (LAM) and relatedspecies, in bodily fluids including but not limited to urine.Heretofore, tests of this nature lacked sensitivity and were notoperable for unprocessed urine samples or for detecting extrapulmonaryTB infections. In particular, we have developed enriched antibodiesraised to antigen from mycobacteria wherein the antibody is enriched byhaving been raised in an environment that maintains antigenically activeantigen. We call the method for producing this first class of enrichedantibody the “direct method,” which is described in further detailbelow.

We have also developed antibody that is enriched by exclusion ofantibodies that recognize relatively inactive antigen. The method forproducing this class of antibodies begins by following the “directmethod” to obtain enriched antibodies, but then also operates byexcluding antibodies that recognize relatively inactive antigen. We callthe method for producing this second class of enriched antibody the“enhanced method,” which is also described in further detail below.

FIG. 8 is a schematic depiction showing the steps involved in practicingan embodiment of the enhanced method. Because the enhanced method buildson the direct method, FIG. 8 also illustrates the direct method, if onestops after the first affinity column.

Below we show how these enriched antibodies of either or both classescan be used to detect pulmonary and extrapulmonary infections of TB in avariety of samples, including but not limited to untreated (i.e.non-concentrated) urine samples. (Other potential sources of sampleinclude sputum, cerebrospinal fluid, blood, tissue, lavages.) In theexamples which follow, the enriched antibodies are raised to an epitopeof lipoarabinomannan (LAM) in an environment which maintains itsantigenic activity.

Prior methods for detecting surface polysaccharides (LAM) usingdifferent body fluids such as serum, urine or sputum have beeninvestigated, but have proven problematic. In serum, the detection ofLAM seems to be disturbed by immune complex formation. Detection of LAMin sputum is possible only in the samples of the patients with pulmonaryTB because extra-pulmonary infections often do not provide sputumcontaining mycobacterial antigens. Prior studies with urine requiredextensive sample processing and manipulation, limiting suchmethodologies in the field. None were effective for diagnosingextra-pulmonary mycobacterial infections such as those on the rise inHIV-positive subjects.

Embodiments of the present invention overcome difficulties in the priorart by providing enriched antibodies that may be used for detectingmycobacterial antigens in a wide range of sample types from a subject.These sample types include sera, blood, sputum, lavages, tissue, andunprocessed, non-concentrated urine, among others.

Lipoarabinomannan (LAM) is a 17500 mol wt lipopolysaccharide specificfor the genus mycobacterium. Lipoarabinomannan is a complexpolysaccharide antigen composed of mannose and arabinose residuesforming a highly branched and complex structure. Despite more than fourdecades of structural studies of polysaccharide antigens ofmycobacteria, those in the art still speak only about fragments of thestructure or structural motifs and composite models. The most recentcomposite model of LAM structure is presented in FIG. 1, below.

As part of the outer cell wall of mycobacteria, LAM is released frommetabolically active or degenerating bacterial cells. It is assumed thatin active TB infection LAM leaks into the circulation, passes throughthe kidneys and can therefore be detected in the urine reflecting thelevel of mycobacterial burden. Since LAM is a carbohydrate antigen withglycosidic linkages for which no human degrading glycosidases exist, theantigen occurs in the urine in intact form.

LAM antigen of mycobacteria is composed of three major structuraldomains: the mannosyl-phospahtidyl-myo-inositol (MIP) anchor, containingvariable number of fatty acids with variable chain length; mannan corepolysaccharide variable in number of mannose residues; and branchedarabinan polysaccharide chains connected to mannan core. Despite manyefforts, the attachment site(s) for arabinan chains on the mannan coreremain unknown. Arabinan polysaccharide chains are capped by mannoseoligosaccharides, consisting of mono-, (α1-2)-di- and(α1-2)-tri-mannosyl units variable in their length (capping motifs).Capping degree is variable from strain to strain and possibly is alsodependent from growth conditions.

Extremely high structural complexity and variability of mycobacterialLAM lead to very complex spectrum of antigenic epitopes. Complexity ofthe selected diagnostic antigen forces us to use affinity purifiedpolyclonal antibody as a main immunoassay reagent. Only use ofpolyclonal antibody allows one to cover the full spectrum of antigenicspecificities potentially associated with LAM present in clinicalsamples. In order to achieve the highest possible assay sensitivity ofsandwich immunoassay, we use the highest concentration ofantigen-specific antibody in the capture zone and also as the labeledantibody. Antigen-specific affinity purification is known to producesuch an antibody.

To prepare the antigen-based affinity column, we developed a process forantigen isolation and coupling to the solid phase support. The processof LAM antigen isolation is based, with some minor modifications, on themethods of isolation of other bacterial polysaccharides described in theliterature and well-known to those in the art, and described below.

Previous LAM-based direct antigen immunoassay described in theliterature used polyclonal antibody purified by antigen-specificaffinity chromatography using a LAM-Sepharose column. The prior artapproach to the synthesis of the affinity matrix was based on thepartial NaIO₄-oxidation of LAM polysaccharide with subsequent couplingto NH₂-Sepharose. Surprisingly, our experiments have shown thatNaIO₄-oxidation reduces antigenic activity of LAM polysaccharide, as canbe seen from the FIG. 2.

Because coupling efficiency of oxidized polysaccharide to NH₂-solidsupport is proportional to the degree of oxidation, we have coupled toSepharose support via functionalized BSA-spacer molecule LAM antigenoxidized with 50 mM NaIO₄. At this level of oxidation LAM polysaccharidestill retains some antigenic activity, as described below, but provideshigh coupling efficiency. Application of the immune serum to suchaffinity matrix resulted in the isolation with high yield of thefraction of rabbit antibody. Testing of such antibody in the plate ELISAimmunoassay format as a capture antibody showed some functionalactivity, but not at the level sufficient to be used in the highsensitivity immunoassay necessary for screening applications usingnon-concentrated urine samples. These data explain results obtained inthe literature previously, where LAM-specific affinity purified antibodywas used, but it was still necessary to concentrate urine samples inorder to detect Lam present in the samples.

Unexpectedly, by changing the LAM coupling chemistry to a mildernon-destructive process, based on polysaccharide activation withcyanogen bromide (CNBr) resulted in the purification of a much betterquality of LAM specific antibody, as can be seen in FIG. 3.

Then, surprisingly, passing the antibody purified on the column withintact LAM (CNBr-activation process) through a column with LAM antigenafter deep, strong NaIO₄ oxidation (see below) produced a relativelysmall fraction of antibody, approximately 7-10% of the applied amount,with very high activity in the LAM-specific direct antigen immunoassay.FIG. 3 shows the efficiency of such antibody as a capture antibody. Whensuch antibody was labeled with horse radish peroxidase (HRP) and used asa labeling antibody, it also demonstrated activity higher than any otherantibody tested or known. The ELISA system based on such antibody hasshown extremely high sensitivity and proven to be useful in testingnon-concentrated urine samples. This enabled us to produce a screeningLAM-specific immunoassay with performance characteristics suitable forrapid screening, in the field, or both pulmonary and extra-pulmonary TBcases, a feat unattainable by others before. Thus, although LAM has beendescribed in the frozen urine of TB patients, the assay for such reportsrequires an extensive sample preparation and therefore is not fieldadapted.

Protocols

In this section we describe protocols suitable for practicing the“direct method” and the “enhanced method” defined above. This discussionis not sorted strictly according to the direct method and the enhancedmethod per se, but describes specifically methods of preparing columnssuitable for use in either or both methods, depending upon the context.FIG. 8 shows a schematic of the direct and enhanced methods.

Isolation of Dry Cells of M. tuberculosis from Freund's Adjuvant

First, allow cells with Freund's Adjuvant to settle for a minimum of 1week at room temperature before use. Remove caps from adjuvant vials andwithout disturbing cells settled on the bottom of the vial, pull off thebulk mineral oil. A small amount of mineral oil may be left in the vialas a precaution to avoid drawing cell precipitate. Discard mineral oiland then mix 6.0 L of ethanol and 6.0 L diethyl ether, and add 5 mLethanol:diethyl ether mixture to each vial.

Next, close the vial, vortex and quickly transfer the suspension into a1-L Erlenmyer flask. Avoid letting cells resettle in the vial duringthis step. When the 1-L Erlenmeyer flask is filled to the 1-L line, letthe cells settle for 1-1.5 hr. Next, gently decant solvent from theErlenmeyer flask into a clean 1-L beaker. Avoid disturbing settled cellsand moving them with solvent. If solvent decanted into beaker is clear,discard it. If a significant amount of cells were decanted with thesolvent, return the decanted solvent to the Erlenmeyer flask and repeatsettling step. Using 20-30 ml aliquots of ethanol: diethyl ethermixture, transfer cells onto a glass sintered filter.

Wash the cells with 500 mL of an ethanol-diethyl ether mixture, thenwash with 200 ml of diethyl ether. Next, air-dry cells on a filter usinga vacuum of 100 mmHg+/−10 (low vacuum) Occasionally mix and homogenizethe cell mass, then cover the filter with a porous material (such as aKim-wipe) and leave in hood until dry (approximately 15 hours, i.e.overnight).

Weigh and record the total weight and calculate the dry weight of thecells. Then tightly seal with rubber lined cap and store at 15-30° C.

Phenol Extraction of Crude LAM Antigen

Place the dry cells of M. tuberculosis into a 250-mL Pyrex media bottleand add warm deionized water to the cells. Vortex and pulse sonicate(˜20 second pulses) the suspension in the ultrasonic water bath untilsuspension is homogeneous.

Phenol extract the cells, then ethanol precipitate and place theprecipitated cells in the refrigerator (2-8° C.) overnight (˜16 hours)to allow the precipitate to settle. Being very careful not to disturbprecipitate, gently draw off the supernatant until about 100 mL ofsupernatant is left covering the precipitate. Gently swirl to mix, thentransfer the remaining suspension into teflon centrifuge tubes andcentrifuge at 12,000 rpm for 20 minutes. Draw off as much supernatant aspossible from all tubes with out disturbing the pellet, add 5 ml ofdeionized water to each tube and, using vortexing and pulse sonication,dissolve pellet in water. Combine all the fractions with the dissolvedpellet and place in a 500-mL flask (Note: do not exceed 1/10 of theflask capacity/volume). Rotary evaporate to minimal volume, but avoidcaramelizing the sample. Redissolve film with approximately 50 mL ofwater and repeat drying and redissolving until sample has been dried 3times. Redissolve in 50 mL of water and lyophilize.

Purification of LAM Antigen by Sephadex G-25 Chromatography

Dissolve 800 mg of crude LAM Ag in 15 mL of 0.25% acetic acid solution.Vortex and sonicate in ultrasonic bath to achieve complete dissolution.Centrifuge in a microcentrifuge at 5000 rpm for 5 min. Collect thesupernatant in a 20-mL glass vial, divide the supernatant into 3 equalparts for separate chromatographic runs, and then gently apply ⅓^(rd) ofthe LAM Ag supernatant collected above onto the chromatographic column.After a volume of 100 mL has flowed through the column, begin collectingfractions. Continue collecting fractions until 350 mL of mobile phasehas passed since the start of chromatography. Cover all fractions andstore at 2-8° C. Rotary evaporate in a 250-mL evaporation flask (novolumes greater than 25 mL). Evaporate to minimal volume, but avoidcaramelizing the sample. Dilute evaporated material in 20 mL of water,sonicate, vortex until complete dissolution and then lyophilize(approximately 8 hours). Scrape dried material with a spatula into atared glass vial and weigh.

The foregoing steps are depicted schematically in FIG. 6.

Coupling LAM Antigen to BSA-Spacer by CNBr Activation.

First, prepare 0.5 M sodium bicarbonate and 1 M potassium carbonatesolutions. Then dissolve 30.0 mg of purified LAM Ag in 1.5 mL ofdeionized water. Use pulse sonication (10-20 sec pulses) and vortexingto completely dissolve the LAM Ag.

Dissolve 300 mg of BSA-hydrazine ligand in 15 mL of deionized water.Pulse sonicate (10-20 sec pulses) and vortex to dissolve completely,then place in microfuge tubes and centrifuge in a microcentrifuge for 10minutes at 10,000 rpm. Using a Pasteur pipette carefully collect andpool the clear supernatant from each tube and transfer into a 20-mLvial. Avoid disturbing any pellet that may form. Add 1.0 mL of 0.5Msodium bicarbonate to the vial and mix well by shaking Add 150 μL ofchilled 1M potassium carbonate to the LAM solution and mix well by briefvortexing. Place obtained solution in ice/water bath.

Prepare 5 mg/mL CNBr in acetonitrile for immediate use and add 180 μL ofthe cyanogen bromide solution to the LAM solution. Mix by vortexing andplace on ice for approx. 15 minutes. Add this solution to theBSA-Hydrazine ligand solution (above) with a Pasteur pipette. Mix welland incubate overnight (16-24 hours), at 2-8° C., in tightly sealedvial.

Coupling of LAM Antigen to BSA-Spacer by NaIO₄ Activation

Dissolve the LAM antigen in 1.25 mL of deionized water in a 3-4-mL vial.Pulse sonicate and vortex to dissolve completely. Prepare a 0.1M sodiumperiodate solution: (in NaOAc buffer, pH 4.0). Add 1.25 mL of the 0.1MNaIO₄ solution to the 1.25 mL LAM solution. Vortex to mix. Cover thevial with aluminum foil; place it on the rocking platform and mix for 1hour+/−5 minutes at ambient temperature.

Dissolve 250 mg of BSA-hydrazine ligand in 12.5 mL of deionized water ina 25-40 mL glass serum vial. Use pulse sonication and vortexing todissolve completely, then centrifuge for approx. 10 minutes at 10,000rpm. Using a Pasteur pipette collect the supernatant from each tube andpool into a 25-40 mL glass vial. Avoid disturbing the pellet. Add 12.5mL of 0.1M sodium phosphate (pH 6.8) to the vial and mix well by briefvortexing.

Coupling Process:

To the BSA-hydrazine solution add the oxidized LAM solution and vortex.Add 100 mg of sodium cyanoborohydride and seal. Sample 10 μL of thefinal solution and dilute with 90 uL 1×PBS buffer (QC solution) andretain for further analysis (LAM concentration will be approximately0.75 mg/mL).

Activation of Sepharose by NaIO₄

Measure an aliquot of suspension of Sepharose 4B-CL corresponding to 80ml of settled gel and transfer onto a sintered glass filter. Wash with500 mL water and drain using low vacuum (approx 300 mmHg) until thegranular structure of the gel surface becomes visible. Avoid formationof the air cracks in the gel layer. Prepare a 0.1 M sodium acetatebuffer, pH 4.0 solution and use to prepare a 30 mM solution of NaIO₄ in0.1 M NaOAc.

Add 250 mL of 30 mM NaIO₄ to the gel and thoroughly mix. Cover themixture with aluminum foil and place at a 45° angle on a rocker platformat medium speed for 1.5 hours±10 minutes at ambient temperature.Transfer to the sintered glass filter and wash with 1 L of water usinglow vacuum (approx 300 mmHg). The activated gel must be prepared withina maximum of 4 hours of use.

Coupling of BSA-LAM ligand to activated Sepharose (For Synthesis offirst and second affinity columns)

Preparation of Matrix

Prepare a 0.1% sodium azide solution in 1×PBS (phosphate bufferedsaline). Measure a suspension of activated Sepharose corresponding to 60ml of the settled gel (or other suitable matrix) and transfer it onto asintered glass filter. Drain gel using low vacuum (300 mm Hg) until thegel packs and granular structure becomes visible, but avoid formation ofcracks on the gel surface.

BSA-LAM Ligand Solution:

In a 250-mL media bottle dilute approximately 17 to 20 mL of thesolution of BSA-LAM ligand to 90 mL with sodium phosphate buffer (pH6.8). Add 90 mg of crystalline sodium cyanoborohydride to the solution.Tightly close the bottle using the supplied plastic cap. Mix well byvortexing briefly. The solution may appear opalescent but there shouldbe no precipitate. Microscopic gas bubbles formed by the sodiumcyanoborohydride may be visible.

Coupling Step:

To the LAM solution prepared above add the drained activated Sepharosegel. Tightly close and thoroughly mix the suspension using gentlevortexing. Incubate for approx 4 hours at 37° C.+/−2° C., mixing (byinversion) the reaction mixture every hour. Add 4.5 mL of 1.5 M Trisbuffer and tightly close cap again. Continue incubating at 37° C.+/−2°C. for approximately 16 hours (overnight).

Transfer the reaction mixture onto a sintered glass filter and collectthe liquid phase into a clean 100-200 mL Bunzen flask by applying lowvacuum (300 mm Hg). Wash the LAM-Sepharose gel on the filter with 400 mlof deionized water and continue washing with 600 mL of 1×PBS

Packing and Storage of Column:

In a 250 mL beaker add 100 mL of 1×PBS to the prepared gel. Stirmanually into a slurry. Pack into a column according to standardprocedures, using 1×PBS. Equilibrate the column with 1×PBS plus 0.1%sodium azide.

Generic Coupling of LAM Ligand to Activated Sepharose (for Preparationof Affinity Columns I and II)

Measure suspension of Activated Sepharose corresponding to 100 ml of thesettled gel and transfer it onto a sintered glass filter. Drain gelusing low vacuum (300 mm Hg) until the gel packs and granular structurebecomes visible, but avoid formation of cracks on the gel surface.Retain drained gel for later use.

BSA-LAM Ligand Solution:

In 250 mL Pyrex media bottle dilute approx. 27.5 mL solution of BSA-LAMligand to 100 mL with the sodium phosphate buffer (pH 6.8). Add 100 mgof crystalline sodium cyanoborohydride to the solution. Tightly closethe bottle using the supplied plastic cap. Mix well by brieflyvortexing. The solution may appear opalescent but there should be noprecipitate. Microscopic gas bubbles formed by sodium cyanoborohydridemay be visible.

Coupling Step:

To the LAM solution prepared above add the drained activated Sepharosegel. Tightly close with the supplied plastic cap. Thoroughly mix thesuspension using gentle vortexing (medium speed) and incubate for approx4 hours at 37° C.±2° C., mixing the reaction mixture (by inversion)every hour. Add 7.5 mL of 1.5 M Tris buffer and tightly close. Continueincubating at 37° C.±2° C. for approximately 16 hours (overnight).

Transfer the reaction mixture onto a sintered glass filter and collectthe liquid phase into a clean 100-200 mL Bunzen flask by applying lowvacuum (300 mm Hg). Wash the LAM-Sepharose gel on the filter with approx800 ml of deionized water. Continue washing with approx 1.2 L of 1×PBS.

Packing and Storage of Column:

In a 250-mL beaker add approximately 160 mL of 1×PBS to the gel above.Stir manually (with spatula/glass rod) into a slurry. Pack into a columnaccording to standard procedures using 1×PBS. Equilibrate the columnwith 1×PBS with 0.1% sodium azide.

The foregoing steps involving use of purified LAM and preparation ofaffinity columns I and II are depicted schematically in FIG. 7.

Isolation of Antibody by Affinity Chromatography-I (the “DirectMethod”).

Prepare the following stock solutions:1 liter of 0.1M glycine buffer and adjust the pH to 2.5 with 1M HCl.1 liter of 3×PBS solution (dilute a 10×PBS stock solution with deionizedwater) and check the pH, and re-adjust to 7.2 to 7.4, if needed, with 1MHCl or 1M NaOH.200 mL of a 1×PBS plus 0.1% sodium azide solution.100 mL of a 0.5 M disodium hydrogen phosphate (Na₂HPO₄) solution

Serum Preparation

Slow-thaw frozen serum in the refrigerator (approx 16 hours/overnight)until completely thawed. Measure sera volume and weigh 2.9 g of sodiumchloride for every 100 mL of serum and add to the sera. Swirl gentlyuntil completely dissolved: the final concentration will be 0.5M NaCl.

Centrifuge (4-8° C.) at ˜8000 g for 20 minutes. Draw off supernatantfrom all centrifuge tubes with Pasteur pipette. Do not to disturb thepellet. Filter supernatant through a cotton-plugged funnel and collectthe filtrate. Collected filtrate should be slightly opalescent, butshould not contain any particulate materials. Place filtered serum inthe refrigerator until the beginning of the affinity chromatographystep.

Serum Application:

Prepare column I (non-modified LAM coupled to column material) for serumapplication by equilibrating with 1×PBS. Adjust the flow rate to 2.0mL/min and continue applying 1×PBS until the baseline remains stable forat least 1 hour. Adjust zero for the recorder and detector as needed.Once the baseline is stable, adjust the flow rate to 0.5 to 0.6 mL/min.and then apply the serum prepared above to the LAM Affinity Column I atthe 0.5-0.6 ml/min flow rate. Collect void volume eluant (it will beapproximately 30% of the column volume). Monitor fractions by UVdetection at 260-280 nm and when an increase in signal occurs, begincollecting serum passed through the column in a 500-1000 mL serum. Afterthe entire volume of serum has been applied to the column, briefly stopthe column flow, apply 3×PBS buffer, and then resume liquid flow.Continue to wash column with 3×PBS until the signal decreases toapproximately 50% of baseline. At this point stop collection of serumand save all collected fractions. Change the flow rate to 2.0 mL/min andcontinue washing the column with 3×PBS until baseline is approximately10-15%. Discard flow-through. Replace 3×PBS buffer with 1×PBS buffer andwash with approx 2-2.5 column volumes at a flow rate of 2.0 mL/min.Discard flow-through.

Elution of Antibodies Step:

Adjust flow rate to 1.0 mL/min. Replace 1×PBS with cold 0.1M Gly-HClbuffer prepared above, and start elution of the adsorbed antibody. Whenthe signal increases rapidly and gains about 10-15% of the full scale,begin collecting eluent column into 15 ml conical tubes placed inice-water bath (0° C.). Collect 5-ml fractions.

Continue collecting antibodies in Gly-HCl buffer until the signal beginsto decrease rapidly. Stop fraction collection when the signal drops tothe signal level of the beginning of collection (10-15% of full scale).Neutralize the collected antibody solution by to each 5-mL fraction 0.5mL of 0.5M Na₂HPO₄ in 0.1-ml increments. The total volume added shouldbe equal to 10% of the fraction volume before neutralization.

Gently mix solution during addition of Na₂HPO₄ buffer and pool theneutralized fractions. Measure the O.D. of antibodies at 280 nm againsta blank containing only 0.1M Gly-HCl buffer and calculate the antibodyconcentration. Place the antibody collected at 2-8° C. for a minimum of3 days to allow crashing and shedding.

Column Care:

Equilibrate the column with 1×PBS until neutral (pH 7). During non-use,equilibrate the column with the 1×PBS plus 0.1% sodium azide solutionand store the column at 4-8° C. until future use.

Dialysis

Centrifuge prepared antibodies at 10,000 G for a minimum of 5 minutes.Transfer the supernatant to 12-14 mol. wt. cut-off dialysis tubing anddialyze against 1×PBS for 2-3 days with a minimum of 4 changes ofbuffer, with a ratio of Ab solution to total volume of ≧1:20. Removeantibodies from dialysis. Measure volume of antibody solution usingglass graduated cylinder. If there is any additional crashing/shedding(in the form of a precipitate) centrifuge the antibody solution again at10,000 G for a minimum of 5 minutes. Measure the O.D. of antibodies at280 nm after blanking the spectrophotometer with 1×PBS buffer. Calculatethe concentration in mg/mL and place for storage at 4-8° C.

Isolation of Antibody by Affinity Chromatography-II (the “EnhancedMethod”) Purification of Highly Specific Antibodies

Apply 1×PBS to the LAM Affinity Column 2 prepared above, (LAM modifiedby strong oxidation, coupled to column material using NaIO₄), at a 2.0mL/min flow rate until the baseline remains stable for at least 15minutes. Adjust the recorder and detector to Zero, as required. Continueto monitor the baseline for the next 30 minutes and once stable, applyantibody to the column. Adjust the flow rate to 0.5-0.6 mL/min and applya volume of antibody, as prepared above, corresponding to ˜100-150 mg ofAb to the LAM Affinity Column 2 using an Econo pump or similar device.Collect void volume eluate (It will be approximately 30% of the columnvolume) at 280 nm. Begin collecting antibodies as the signal increasesto about 10-15% above baseline in a clean serum bottle. When the totalantibody volume has been applied, briefly stop the liquid flow, apply1×PBS buffer and resume liquid flow at 0.5-0.6 mL/min. Continue tocollect material flowing through column at 280 nm. When the signal dropsto 10-15% above the start of collection (30-50% above baseline), stopcollecting the solution.

Measure the O.D. of highly specific antibodies at 280 nm after andcalculate the antibody concentration. Immediately place antibodysolution at 4-8° C. for temporary storage.

Column Wash

Continue to wash the column with 1×PBS at a flow rate of 2.0-2.5 mL/min.Pass minimum 3 column volumes of 1×PBS. Elute material absorbed ontocolumn with cold 0.1M Gly-HCl buffer, prepared above. Collect materialeluted in glass vials. When the monitor/signal drops to ˜10-15% ofbaseline, stop collection. Neutralize the collected Antibody solution byadding 10% of total volume of 0.5M sodium phosphate, prepared above, byadding in 0.5 mL increments. Measure the O.D. of antibodies at 280 andcalculate the antibody concentration. Immediately place the collectedantibodies solution at 4-8° C. and retain until the analysis ofantibodies collected in step above is complete. If the concentration ofantibodies above is less than 0.3 mg/mL, concentrate.

Wash the column with a minimum of 3 column volumes of 1×PBS at a 2.0-2.5mL/min flow rate. Wash the column again with 1 column volume of 1×PBSplus 0.1% sodium azide, and store at 4-8° C. until future use.

The foregoing steps showing isolation of enriched antibodies fromaffinity columns I and II using the direct and enhanced methods aredepicted schematically in FIG. 8.

ELISA Plate Coating Process. Set-up of the Moduline 300 System.

The Ab coating must be completed within maximum 8 hours from end ofpreparation of the coating solution M815. The Antibody coating solutionmust be kept in on ice (0° C.) during the coating process.

Step One

Pre-weigh and inspect empty plates and discard any broken plates.Dispense 100 μl of MTB-LAM specific Ab coating solution into each wellof each strip plate using a Moduline 300 System. Visually check all the96 wells in each plate for uniformity of well filling during coatingprocess. Save unused Ab solution and store at (2-8° C.) until thecomplete lot of plates are processed and passed for use. Stack plateswith dispensed Ab in stacks of 10 plates each and cover the top platewith an empty plate used as a cover. Label each stack cover plate from 1to 18. Refrigerate the stacked plates at 2-8° C. and incubate overnight(14-18 hrs).

Step Two:

Set-up of the Moduline 300 System to perform 3-times wash cyclesfollowed by immediate dispense cycle of 312 uL Block Solution. BlockSolution must be used within maximum 24 hours from end of preparation.Remove plates from refrigerator and remove the covering plates fromstacks as they are being placed on the Moduline and place them aside.Set the timer for 6 hours. Set blocked plates coming from the conveyor,on sequentially numbered trays and block for 5 to 6 hours at ambienttemperature(20-28° C.).

Place the plates on trays in the Drying Chamber and incubate at 20-23°C. and 20-22% relative humidity for 24-72 hr. Remove dry plates fromdrying chamber.

MTB-Ab Preparation for Conjugation to HRP

(MTB-Ab Solution Preparation should be Performed at Least 7 Days BeforeConjugation Procedure.)

Dialysis:

Dialyze the necessary amount of MTB-LAM-Ab solution against 1×PBS forminimum 48 h with minimum 4 changes, at 2-8° C. Use dialyzing tubingwith MWCO 12-14,000. After dialysis centrifuge Ab solution at 12,000 rpmfor 10 min. and carefully aspirate supernatant into the 15 ml graduatedcentrifuge tube.

Measure optical density of Ab solution after dialysis at 280 nm andCalculate Ab concentration after dialysis. If Ab solution after dialysishas OD_(280 nm)>2.8, make a 1:7 dilution of Ab solution in 1×PBS.

Concentration:

Prewash an Amicon Ultrafree-15 centrifugal filter device with 1×PBS.Place approx. 15 mL of 1×PBS solution into device and centrifuge at 3500rpm for approximately 5 min. Discard all the solution from device units.Concentrate the above Ab solution after dialysis with Ultrafree-15centrifugal filter devices to 4.5-5.5 mg/ml by centrifugation onBench-top centrifuge (bucket rotor) at 3500 rpm for approx. 5 min.×3.Carefully aspirate the concentrated Ab solution from the filter unit ofthe Amicon device into a 15 mL tube. To maximize recovery, removeconcentrated sample immediately after centrifugation and resuspendconcentrate volume several times with a pipette to ensure proper mixingbefore Ab aspiration.

Centrifuge the concentrated Ab solution at 10000 rpm for approx. 15 min.and aspirate the Ab supernatant into a 15 mL tube. Measure the OD₂₈₀ ofAb solution at 1:20 dilution in 1×PBS and calculate concentration of Ab.

Sample 0.1 ml of Ab solution for ELISA analysis. Store at 2-8° C.

MTB-LAM-Ab-HRP Conjugate Preparation

Wash all glass vials and stir bars for conjugation steps and glass vialsfor conjugate storage with H₂SO₄ solution and thoroughly rinse them withtap water and deionized H₂O.

Preparing the Sephadex G-25 Column for Chromatography:

Obtain a column (1.5×30 cm) with approximately V=50 ml packed withSephadex G-25 (Fine). Pack the column as described above. Set thefollowing chromatography conditions to equilibrate the column with the 1mM Sodium

-   -   Acetate Buffer, pH 4.4.    -   UV Monitor wavelength for 280 nm    -   Monitor Sensitivity: 0.2 OD    -   Chart recorder speed: 2 mm/min.    -   Pump Flow rate for column washing: 60 ml/h        Wash the column with approx 100-150 ml of 1 mM Sodium Acetate,        pH 4.4 and adjust the UV monitor baseline to 0-position. Make        sure that established base line is stable for approx. 30 min.        Calculate the amount of MTB-LAM-Ab solution necessary for        conjugation and centrifuge Ab at 12,000 rpm for approx. 10 min.        Carefully aspirate the Ab supernatant into a clean glass vial.        Measure the OD_(280 nm) of Ab solution at 1:20 dilution in 1×PBS        and Calculate concentration of the undiluted Ab solution. Store        the Ab solution at 2-8° C. until use.        Oxidation of HRP (Horse Radish Peroxidase) with NaIO₄:

Weigh 8 mg of HRP in V-shaped glass vial. Add 2.0 ml of deionized H₂O.Gently stir the solution for approx. 2-3 min. until all the HRP hasdissolved. Make sure there are no undissolved HRP particles on the glassvial walls left.

Prepare a fresh solution of 0.1 M NaIO₄, pH 4.4 for use within a maximumof 5 minutes and protect from light. Add 0.4 ml of 0.1 M NaIO₄ to theHRP solution prepared above, while stirring. Cover the vial withaluminum foil to protect the mixture from light. Incubate the mixturefor 20 min. with stirring at ambient temperature. Add 4 drops ofethylene glycol to the reaction mixture and stir for approximately 2min.

Chromatography and Concentration of Oxidized HRP:

Immediately after completing the above step purify the oxidized HRP bygel-filtration on Sephadex G-25 (Fine) column. Set the pump flow ratefor sample elution to approximately 50 ml/h. Carefully apply the totalvolume of the oxidized HRP prepared above onto the dry gel bed but takecare not to disturb the gel bed. Do not over dry gel. Collect alloxidized HRP (colored solution) into one 15 ml tube. −1^(st) peak on thechromatography Chart (OD_(280nm)>0.05). After chromatography iscompleted, empty the column of Sephadex G-25 and discard the gel andrecord the volume of HRP solution after chromatography.

Concentrating Oxidized HRP

Prewash Ultrafree-15 centrifugal filter devices with 1 mM SodiumAcetate, pH 4.4 with approximately 15 mL of 1 mM Sodium Acetate, pH 4.4,and centrifuge the filter unit for approx. 5 min. at 3500 rpm using abench-top centrifuge (bucket rotor). Then discard all solutions from thefilter unit. Immediately after chromatography, concentrate the oxidizedHRP solution (from above) to approx. 2±0.2 ml with an Ultrafree-15centrifuge filter unit (Biomax-10K membrane) by centrifugation at 3500rpm for approx. 5 min. Carefully aspirate the concentrated HRP solutionfrom the filter unit of the device into the clean glass vial, measureand record the volume, and store at 2-8° C.

Conjugation HRP to MTB-LAM-Ab:

Calculate the amount of MTB-LAM-Ab solution necessary for conjugation toHRP. Place the MTB-LAM-Ab (from above) into a V-shaped glass vial withtriangular stir bar, without leaving drops of the Ab solution on thevial walls. Add ½ volume of oxidized HRP solution (above) to theMTB-LAM-Ab solution, cover the vial with aluminum foil to protectreaction mixture from the light and stir reaction mixture in the glassvial for 30 min at room temperature. Avoid foaming.

Add 1 M Carbonate-HCl, to pH 9.5 and stir at room temperature for twohr. Protect from the light and avoid foaming.

Prepare 4 mg/ml Sodium Borohydride (NaBH₄) immediately before use andprotect from the light with the aluminum foil. Immediately add thecalculated amount of NaBH₄ required to the MTB-LAM-Ab solution preparedabove, and incubate the reaction mixture at approx. 2-8° C. for 2 hr.Dialyze reaction mixture against 1×PBS for minimum 48 h at 2-8° C. witha minimum of 4 buffer changes at 8-16 hours intervals. Use 12-14 kDacut-off dialyzing tubing for dialysis.

Conjugate Storage and Analysis:

After dialysis, centrifuge the conjugate solution at 4000 rpm forapprox. 4 min. Carefully withdraw supernatant and place conjugatesolution into the clean 6 ml glass vial. Measure 18 ml of GardianPeroxidase Conjugate Stabilizer/Diluent into the 50 ml glass bottle withmagnetic stir bar. Add 2 ml of MTB-LAM-Ab-HRP conjugate and stir themixture for approx. 10 min. Store at 2-8° C., and protect from light.

The foregoing steps relating to MTB conjugate preparation are depictedschematically in FIG. 9.

Results

Below we present data from the evaluation of a direct antigen ELISAwhich detects LAM in unprocessed, non-concentrated urine using the“direct method” for enriched antibody production. (It is believed thateven better data will result by using enriched antibodies produced usingthe “enhanced method” described above.) The studies producing these datawere carried out in the Mbeya region that is located in the Southwesternhighlands of Tanzania in collaboration with the Regional TB and LeprosyProgramme and the Mbeya Medical Research Project (MMRP). In the MbeyaRegion approximately 3,500 new TB cases are diagnosed annually andtreatment is conducted according to the national DOTS strategy.Initiation of every therapy is initiated at a central facility at theMbeya Referral Hospital. The TB cure rate was 72.3% in 2002. The aim ofthe study was to evaluate the performance of a commercially availableLAM-capture ELISA in clinical practice and to compare the results withthe gold standard for TB diagnosis: Sputum microscopy, TB-culture, chestradiography and clinical investigation.

Material and Methods

LAM-ELISA Description

The MTB-ELISA direct antigen sandwich immunoassay (MTB-ELISA, Chemogen,So. Portland, Me., USA) is a LAM-ELISA similar to an assay developed byothers. The immune sera were harvested from white New Zealand rabbitsthat were immunized with inactivated whole cells of M. tuberculosisH37Rv. Polyclonal LAM-specific antibodies were isolated by affinitychromatography using immobilized LAM as a ligand. The test kit consistsof an 96-well ELISA plate pre-coated with LAM-specific antibody, blockedand sealed in a plastic pouch with desiccant; a vial with LAM-specificHRP-conjugated LAM-specific polyclonal antibody; a vial with TMB(3,3′,5,5′-tetramethylbenzidine) single component chromogenic substrate;a vial with the negative control solution, and three vials withcalibrators corresponding to 0.5 ng/ml, 1.5 ng/ml and 4.5 ng/ml of LAMin urinary samples. Urine samples were considered positive in the ELISAwhen the obtained optical density at 450 nm was at least 0.1 abovesignal of the negative control (>2SD).

A patient urine sample of 0.1 ml is placed in duplicates on the ELISAplate, incubated for 1 hour and washed with 0.05% Tween-20/PBS (PBST)solution. 0.1 ml of LAM-specific HRP-conjugate are added. After 1 hourincubation the plate is washed with PBST solution and 0.1 ml of TMBsubstrate are added. After 10 minutes of incubation time the substratereaction is stopped by adding 0.1 ml of 1M H₂SO₄ and the colordevelopment is read at 450 nm.

In other embodiments, the specific isoform of lipoarabinomannan (LAM)determined to contain the antigenic activity is used to generate highlyspecific, highly pure polyclonal antibodies for use in the detection ofmycobacterium lipoarabinomannan in the urine of patients to be screenedfor active tuberculosis, using protocols similar to that describedabove. The antigenically active isoform of LAM was identified usingselective oxidation of LAM, wherein two isoforms were readilyidentifiable and distinguishable (data not shown). One containedportions sensitive to high concentrations of sodium periodate (NaIO₄)such that at high concentrations of sodium periodate the serologicalactivity of the LAM was destroyed. The other isoform maintainedserological activity, even when subjected to high concentrations ofsodium periodate. A comparison of two methods of oxidation of LAM, usingeither mild oxidizing agents or low concentrations of NaIO₄ preservedthe antigenic activity of the LAM. Oxidation by high concentrations ofNaIO₄, however, resulted in loss of antigenic activity of the LAM.

Therefore, only LAM activated with CNBr, or oxidized with mild oxidizingagents or low concentrations of NaIO₄ is used to generate highlyantigenic LAM for use in the preparation of highly specific, highly purepolyclonal antibodies for use in detecting LAM in urine samples fordiagnosing TB in patients of interest.

These results are completely unexpected compared to the detectionmethods disclosed by Svenson et al. (see e.g. WO97/34149) which usedonly high concentrations of NaIO₄ to oxidize the mycobacterial LAM, andconsequently destroyed antigenic activity of the LAM used to generatedthe antibodies. Not knowing that there was more than one isoform of theLAM to be detected, it was not possible in the earlier disclosure toprepare highly specific antibodies to the antigenically active form ofLAM, because no one prior to these studies even knew that a separateisoform existed that contained the antigenic activity, or that suchactivity was lost during standard means of oxidation, namely, treatmentwith high concentrations of NaIO₄.

Clinical Site Description.

Within eight weeks 242 suspected TB patients were recruited at theoutpatient departments of 5 clinical centers in Mbeya, Tanzania. Thestandard protocol of investigation included clinical assessment, chestradiography, ESR, white blood cell×count and HIV test, 3×AFB staining(Ziehl Neelson) of sputum at day 1, 2 and 3, 2 sputum culture onLoewenstein Jenssen medium and LAM-ELISA in urine and serum.

All patients had clinical signs of TB (cough >4 weeks, night sweats,weight loss, loss of appetite). One hundred thirty-seven of these hadlaboratory confirmed pulmonary TB (PTB), 9 had high radiologicalsuspicion of PTB (pleural effusions or enlarged hilar lymph nodes), and8 showed clinical and radiological signs of military TB. Consentingpatients were tested for their HIV status and 70% were confirmed asHIV-positive. Data were handled confidentially. The study was approvedby the local Institutional review board and the national ethicalcommittee of the Republic of Tanzania.

All laboratory procedures were performed in the laboratory facilities ofthe Mbeya Medical Research Project.

Microscopy and Culture of Sputum Samples

Ziehl Neelson staining and microscopy was done by an experienced andwell qualified lab technician. After decontamination sputum samples werecultured on Loewenstein Jenssen medium in duplicates. Cultures wereexamined weekly for growth for 8 weeks.

Urine Specimens

From each patient 30 ml of urine were collected in a sterile plasticcontainer, which was labeled with the code number of the respectivepatient's data form. 100 μl of fresh and unprocessed urine was added tothe wells of the ELISA plate in duplicate. Negative controls, low,medium and high positive controls were also added to each plate induplicates. Specimens were processed within 24 h and then stored at −20°C. for future testing in Germany.

Control Groups from Tanzania and USA

Urine samples of 23 staff members of the Mbeya Referral Hospital, of 20staff members of Chemogen, Inc. and of 200 patients from 2 clinics inNew York were tested in the LAM ELISA. All of them appeared healthy inclinical examination and did not have any signs of respiratoryinfections.

Results

Preclinical Evaluation of the ELISA System.

FIG. 4A shows the dose response curve using different concentrations ofLAM in urine, wherein solid circles represent ELISA results using LAMfrom M. tuberculosis, and open circles represent the control ELISAresults. The optimal cut off value was defined according to this curveas LAM concentration producing an optical density (OD) exceeding OD ofnegative control by 0.1 OD, that corresponds to more than 2 standarddeviations above the signal of the negative control sample. All sampleswith an optical density above this cut off were considered as ELISApositive. The cut off was equal to approximately 0.25 ng/ml of LAM inuntreated fresh urine.

The MTB-ELISA was evaluated for cross-reactivity with other species andgenera of various Gram-positive and Gram-negative bacteria typical forurinary tract infections and bacterial pneumonia. None of the testedspecies has shown any reactivity in the evaluated LAM-ELISA system evenat the highest tested concentrations, as can be seen by comparing theELISA results for M. tuberculosis (open triangles) with the ELISAresults for other bacterial species tested (solid triangles, open andsolid diamonds, open and solid circles) depicted in FIG. 4B. An analysisof whole cells of various species of mycobacteria in the LAM-ELISAsystem shows cross-reactivity with all tested species of mycobacteria(M.) (FIG. 4C), however, M. tuberculosis H37Rv and M. bovis are detectedmost sensitively. Both species are very close from the immunochemicalstandpoint, but M. bovis is rarely a cause of mycobacterial infection inhumans.

Study Participant Data

According to Table 1 the 242 TB suspects were divided into 3 majorcategories: (1) pulmonary TB patients with confirmed microscopic and/orculture diagnosis, (2) patients with typical clinical and radiographicsigns and (3) patients with clinical symptoms of TB, that were notconsidered TB patients as all available diagnostic tools (radiography,sputum microscopy and culture) were negative.

Group one included 137 patients that had a laboratory confirmedpulmonary TB. 132 were confirmed by Loewenstein Jenssen culture and fivehad a negative culture but positive AFB-stain. Out of the 132 culturepositive cases 62.12% were AFB positive.

Group two comprised an additional 17 patients that were enrolled intothe DOTS therapy program based on radiographic and clinical findings(Table 1). The 88 patients of group three were sputum negative and didnot present specific radiological signs of pulmonary TB and weretherefore not enrolled in the DOTS program.

The mean age of the participants was 34 years. The female male ratio was41:59. The overall HIV prevalence among the 223 patients that agreed tobe tested for HIV was 69.1% (see Table 2). The HIV prevalence was 73.2%among patients with and 60.8% among patients without confirmed TB.

Clinical Evaluation of the ELISA

Of the 137 patients with confirmed pulmonary TB (culture or AFBpositive) 111 were LAM-ELISA positive (sensitivity 81.02%) for thepredefined cut off (optical density (OD) of negative control+0.1). Themean OD increment (=absolute mean OD−OD of negative control) for thesmear and culture positive group (82) was 0.604. For smear negative andculture positive cases (50) the mean OD increment was 0.293 and forsmear positive, but culture negative cases (5) 0.249.

Of the 17 patients in group two that were culture and AFB negative, buthad typical radiological and clinical signs for TB 13 (76.47%) had apositive LAM-ELISA test results with a mean OD increment of 0.183. 13(76.47%) of them were HIV positive.

The remaining 88 patients that came to the special TB clinic withclinical signs suggestive of pulmonary TB were culture and AFB negativeand had no specific radiographic findings for TB. Of these 13 (14.77%)had a positive LAM-ELISA test (mean OD increment 0.184).

Based on the known concentration in the low, medium and high positivecontrol that were included on each plate, it was possible to determinethe approximate LAM concentration of each urine sample based on the ODvalue of the ELISA. Whether the LAM concentration correlates to theindividual burden of tubercle bacteria was assessed in AFB positivepatients. While patients with a low density of tubercle bacteria inmicroscopy (AFB+) had a mean LAM antigen concentration of 0.93 ng/ml inthe urine, patients with an intermediate density of acid fast bacilli(AFB++) had a mean antigen concentration of 1.74 ng/ml in their urineand AFB+++ patients 2.02 ng/ml (FIG. 5). The later value is lower thanthe real concentration of LAM in urine of AFB+++ patients because theELISA reader used in the Tanzania lab could not read signals above twocorresponding to about 4 ng/ml.

The HIV serostatus did not influence the sensitivity of the LAM-ELISA inconfirmed pulmonary TB patients. Of 124 patients with known HIVserostatus and positive TB culture and/or AFB stain 73 of 89 HIVinfected patients (82.0%) were positive in the LAM-ELISA compared to 26out of 35 uninfected individuals (74.3%). Similarly, the sensitivity ofthe AFB was not compromised by HIV serostatus. The sensitivity was 61.2%and 58.8% in HIV infected and negative individuals, respectively.

The specificity of the assay was assessed using the urine of healthyTanzanian and US volunteers. The urine of 23 healthy hospital staffmembers of Tanzanian origin was analyzed. None of the samples was testedpositive in LAM-ELISA (−0.047 mean relative OD, specificity 100%). Urinesamples of 220 healthy volunteers from US were collected and analyzed.All but 4 had an optical density below the cut off 0.1 (specificity98.18%).

Discussion

The classical tools for the diagnosis of TB, sputum culture and smearmicroscopy, have obvious limitations. Both methods only detect cases ofopen pulmonary TB. This significantly impairs the possibility of thedetection of all cases of active TB regardless of the organmanifestation. Therefore multiple new methods have been evaluated in thepast that could supplement the classic tools, especially in resourcepoor settings. The criteria that were set for such a new assay are a) ahigher sensitivity than microscopy, b) comparable specificity, c) alimited additional work load, d) the possibility to diagnosesputum-negative TB and e) a sensitivity that is not impaired by HIVco-infection.

In this first evaluation, the sensitivity of the LAM-ELISA (81% ofculture positives) was superior to AFB-stain (69%). Sensitivity can befurther improved by concentrating fresh urine, which would howeverresult in an additional effort for a lab technician. The detection rateof the LAM-ELISA for cases with radiological confirmed military TB(87.5%) as well as for sputum negative cases with typical radiologicalsigns of pulmonary TB (67%) was encouraging, although the case numberswere not high enough to allow a final conclusion. For healthyindividuals the specificity of the ELISA was high (98.18% in US and 100%in Tanzania). HIV co-infection in culture positive TB cases did notinfluence the sensitivity of the LAM-ELISA.

In comparison to previous published results of the LAM-ELISA the newtest detects LAM at lower concentrations (0.2 ng/ml) than former tests.The sensitivity of the new test was 82.9% (of AFB+) for unconcentratedand fresh urine compared to a sensitivity of 81.3% for the previous testusing processed and frozen urine. The test specificity was 98.36% inthis study compared to 86.9% in the previous study.

The limitation of this cross sectional TB study was the fact that acertain proportion of TB suspected patients remained ambiguous in termsof their TB status (group 2 and 3). To acknowledge this problem we havecreated three major categories for analysis: Group 1: laboratoryconfirmed TB, Group 2: clinically and radiological diagnosed TB, Group3: no laboratory or radiological proof of TB. While we are confidentthat participants in category 1 are true TB cases, we cannot excludethat category 2 and 3 contain some wrongly categorized patients. Wetherefore excluded them from our sensitivity and specificitycalculation. Diagnosis of TB often requires the longitudinal follow-upof patients. Especially sputum negative patients with unusualradiological features would have needed several follow-up consultationsin order to re-question their TB status. In a longitudinal studyclinical as well as diagnostic reevaluation and TB treatment outcomewould have given important additional information to classify group 2and 3 in TB and non TB patients.

Of major interest is the question if there is a quantitative correlationbetween the bacterial burden of M. tuberculosis and the amount of LAMdetected in urine. The only way to address this question in a crosssectional study format was to correlate the AFB sputum staining scorewith concentration of LAM in urine. As shown in FIG. 5 there was anobvious positive correlation of antigen concentration in urine andtubercle bacteria density in sputum. Such a correlation opens up severaladditional applications for the LAM assay. The monitoring of treatmentsuccess and the early recognition of relapses after completion oftreatment are of immediate practical relevance. The combination of asensitive urine assay with the capacity to detect extrapulmonary andAFB-negative TB renders the LAM assay a potent tool in an environmentwith a growing prevalence of extrapulmonary forms of TB and pulmonaryforms with atypical clinical symptoms. The LAM-ELISA could not only beused for the diagnosis of patients with clinical symptoms, but also forscreening HIV positive patients and other high risk groups. Early casedetection of active TB and effective treatment are the two pillars in asuccessful fight against TB. To further explore the role of the LAMassay in this fight we are currently planning several prospective andmulticenter studies.

In summary, the LAM-ELISA can be easily integrated in the routinediagnostic procedures of laboratories of both, developed and developingcountries. It is an easy to use and robust assay. Completion of theELISA requires only 2½ hr and many samples can be analyzed at the sametime. As the antigen Lipoarabinomannan is stable, it was possible tokeep the urine refrigerated for 3 days without significant drop inoptical density. The newly developed MTB-ELISA for detection of LAM inunprocessed urine has the potential of a screening test to be used alsounder field conditions in developing countries.

TABLE 1 Analysis of urinary LAM excretion in the 242 patients coming tothe OPD with clinical suspicion for TB and the 243 clinically healthycontrols. Study Group TB Diagnosis Participants LAM+ LAM− 1: LJ+ and/orAFB 137 111 (81.02%) 26 Laboratory LJ+ and AFB+ 82 68 (82.9%) 14 (17.1%)Confirmed TB Only LJ+ 50 38 (76%) 12 (24%) Only AFB+ 5 5 (100%) 0 (0%)2: Military TB 8 7 (87.5%) 1 (12.5%) Clinically and Pleural effusion or9 6 (67%) 3 (33%) Radiologic-ally enlarged hilar lymph diagnosed TB 3:Only clinical signs of 88 13 (14.77%) 75 (85.23%) No laboratory or TB.radiological proof of TB No enrollment in DOTS 4: No clinical signs ofTB 243 4 (1.64%) 239 (98.36%) Negative Control Group The cut off valuefor LAM-ELISA positivity is 0.1 above the mean optical density of thenegative control on the plate. += positive.

TABLE 2 Proportion of HIV positive patients in the different groups.HIV+ All TB suspects 69.1% Culture+ (119)  71.% AFB+ (77) 72.7% EXPTB(17) 76.47%  223 of 242 patients consented to be tested for HIV.

1. An enriched antibody population highly specific for an antigen of asurface polysaccharide from a mycobacterium.
 2. An enriched antibodypopulation according to claim 1, wherein the antibody is enriched byhaving been raised in an environment that maintains antigenically activeantigen.
 3. An enriched antibody population according to claim 1,wherein the antibody is enriched by exclusion of antibodies thatrecognize relatively inactive antigen.
 4. An enriched antibodypopulation according to claim 2, wherein the antibody is enriched byexclusion of antibodies that recognize relatively inactive antigen. 5.An enriched antibody population according to claim 1, wherein themycobacterium is Mycobacterium tuberculosis.
 6. An enriched antibodypopulation according to claim 5, wherein the surface polysaccharide islipoarabinomannan (LAM).
 7. A process for producing an enriched antibodyhighly specific to an antigen of a mycobacterium, the processcomprising: raising and isolating antibody to antigen from mycobacteria;and separating from the isolated antibodies that population ofantibodies which is specific to relatively inactive antigen to produceisolated enriched antibody.
 8. A process for producing an enrichedantibody specific to an antigen of a mycobacterium, the processcomprising: isolating antigen from mycobacteria under conditionsmaintaining antigenic activity; and raising antibodies to the isolatedantigen while maintaining its antigenic activity.
 9. A process forproducing an enriched antibody highly specific to an antigen of amycobacterium, the process comprising: applying sera from a mammalinoculated with mycobacteria to a first affinity matrix prepared withisolated antigen from mycobacterium such that antibody specific to theisolated antigen is retained by the first affinity matrix; isolatingantibody specific to the isolated antigen from the first affinitymatrix; applying the isolated antibody to a second affinity matrixprepared with modified antigen from mycobacterium such that antibodyspecific to the modified antigen is retained by the second affinitymatrix, wherein the modified antigen has been treated with an agent todeactivate it relative to the isolated antigen; isolating enrichedantibody specific to the isolated antigen by collecting effluent fromthe second affinity matrix, so that the enriched antibody is more highlyspecific and displays higher sensitivity to mycobacterium antigen thannon-enriched antibody.
 10. The process of claim 9, wherein themycobacterium is Mycobacterium tuberculosis.
 11. The process of claim10, wherein the surface polysaccharide is lipoarabinomannan (LAM). 12.The process of claim 9, wherein the agent is sodium periodate.
 13. Theprocess of any of claims 7-9, wherein the surface polysaccharide isisolated from Freund's adjuvant.